Method of treating HIV-1 infection utilizing a multiepitope T cell immunogen comprising gag, pol, vif and nef epitopes

ABSTRACT

The present invention relates to novel immunogens based on overlapping peptides (OLPs) and peptides derived therefrom useful for the prevention and treatment of AIDS and its related opportunistic diseases. The invention also relates to isolated nucleic acids, vectors and host cells expressing these immunogens as well as vaccines including said immunogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/374,334, with a 35 U.S.C. § 371(c) date of Jul. 24, 2014, which is anational phase entry of International Application No. PCT/EP2013/051596,filed Jan. 28, 2013, which claims foreign priority to EP Application No.12382031.8, filed Jan. 27, 2012, each of which is hereby incorporated byreference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted Sequence Listing(38340010003_ST25.txt; Size: 43,571 bytes; and Date of Creation: May 1,2018) filed with the application is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to novel immunogens based on overlappingpeptides (OLPs) and peptides derived therefrom. It also relates toisolated nucleic acids expressing these immunogens as well as vectorsand cells comprising such nucleic acids. The compounds of the presentinvention are useful as vaccines, particularly for the prevention andtreatment of AIDS and opportunistic diseases.

BACKGROUND OF THE INVENTION

HIV infection induces strong and broadly directed, HLA class Irestricted T cell responses for which specific epitopes and restrictingHLA alleles have been implicated in the relative in vivo control. SeeBrander C, et al., Current Opinion Immunol. 2006; 18:1-8. While the bulkof the anti-viral CTL response appears to be HLA-B restricted, therelative contribution of targeted viral regions and restricting HLAmolecules on the effectiveness of these responses remains obscure. SeeKiepiela P, et al., Nature 2004; 432:769-775 and Ngumbela K, et al.,AIDS Res. Hum. Retroviruses 2008; 24:72-82.

In addition, the role that HIV sequence diversity plays in the in vivorelevance of virus-specific T cell immunity is unclear, as functionalconstraints of escape variants, codon-usage at individual proteinpositions, T cell receptor (TCR) plasticity and functional avidity andcross-reactivity potential may all contribute to the overalleffectiveness of a specific T cell response. See Brockman M, et al., J.Virol. 2007; 81:12608-12618 and Yerly D, et al., J. Virol. 2008;82:3147-3153. Of note, T cell responses to HIV Gag have mostconsistently been associated with reduced viral loads in both, HIV cladeB and clade C infected cohorts. See Zuñiga R, et al., J. Virol. 2006;80:3122-3125 and Kiepiela P, et al., Nat. Med. 2007; 13:46-53.

However, none of these analyses assessed the role of responses toshorter regions of the targeted protein(s) that may induce particularlyeffective responses. In addition, it is unclear whether the relativebenefit of Gag is due to any other specific characteristic of thisprotein, such as expression levels, amino acid composition andinherently greater immunogenicity. It is thus feasible that proteinsubunits outside of Gag and within these, specific short epitope-richregions could be identified that: i) induce responses predominantly seenin HIV controllers and ii) which would be detectable in individuals ofdiverse HLA types, not limited to individuals expressing allelespreviously associated with effective viral control.

While some of the earlier studies have indeed controlled for a potentialover-representation of Gag-derived epitopes presented on “good” HLAclass I alleles, concerns remain that a purely Gag-based HIV vaccinemight mainly benefit those people with an advantageous HLA genotype andwill not take advantage of potentially beneficial targets outside ofGag. See Kiepiela, 2007, supra and Honeyborne I, et al., J. Virol. 2007;81:3667-3672. In addition, CTL escape and viral fitness studies havelargely been limited to Gag-derived epitopes presented in the context ofrelatively protective HLA alleles such as HLA-B57 and -B27. SeeSchneidewind A, et al., J. Virol. 2007; 81:12382-12393 and Leslie A, etal., Nat. Med. 2004; 10:282-289. The available information may thus notprovide relevant information for immunogen sequences designed to protectthe genetically diverse majority of the human host population.

Furthermore, many studies have focused on immunodominant targets only,despite some recent studies in HIV and SIV infection demonstrating acrucial contribution of sub-dominant responses in in vivo viral control,among them targets located outside of Gag. See Frahm N, et al., Nat.Immunol. 2006; 7:173-178 and Friedrich T, et al., J. Virol. 2007;81:3465-3476. Together, the current view on what may constitute aprotective cellular immune response to HIV is thus quite likely biasedtowards a focus on immunodominant responses and on responses restrictedby frequent HLA class I alleles and HLA alleles associated with superiordisease outcome. Therefore, the development of HIV vaccines is limitedin part by the lack of immunogens capable of inducing a broad immuneresponse. The present invention addresses the design of such immunogens.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an immunogenic polypeptidehaving an amino acid sequence comprising the sequences SEQ ID NOs 1-16or variants of said SEQ ID NO:1-16, wherein each of said variants has alength of at least 8 amino acids, with the provisos that said amino acidsequence does not comprise any sequence stretches derived from the HIVgenome of a length of 8 or more amino acids other than an amino acidsequence according to any of SEQ ID NOs 1-16 or the variants thereof.

In a second aspect, the invention relates to an immunogenic polypeptidehaving an amino acid sequence comprising at least one sequence selectedfrom the group consisting of the SEQ ID NOs 1-16 or variants thereofwherein said variant has a length of at least 8 amino acids, with theprovisos that:

i) said immunogen amino acid sequence does not comprise any sequencestretches derived from the HIV genome of a length of 8 or more aminoacids other than an amino acid sequence according to any of SEQ ID NOs1-16 or a variant or a fragment thereof, and

ii) when the immunogen comprises only one sequence selected from thegroup consisting of SEQ ID NOs 1-16, then this sequence is not selectedfrom the group consisting of SEQ ID NO: 3, 5, 6 and 16.

In further aspects, the invention relates to nucleic acids encoding forthe immunogens of the first aspect and second aspects, and to expressioncassettes, vectors, a viruses and cells comprising said nucleic acids.

In another aspect, the present invention relates to a vaccine comprisingan immunogenic polypeptide according to any of claims 1 to 11 and one ormore adjuvants.

In another aspect, the present invention relates to the immunogenicpolypeptide, the nucleic acid, the expression cassette, the expressionvector, the virus or the cell of the third aspect, or the compositionvaccine for use in medicine.

In another aspect, the present invention relates to the immunogenicpolypeptide, the nucleic acid, the expression cassette, the expressionvector, the virus or the cell of the third aspect, or the compositionvaccine for use in the prevention or treatment of an HIV infection or adisease associated with an HIV infection.

In another aspect, the present invention relates to a kit comprising theimmunogen of the first and/or second aspects, the nucleic acid, theexpression cassette, the expression vector, the virus or the cell of thethird aspect, or the composition of the fourth aspect.

Deposit of Microorganisms

The plasmid 298H GMCSF-HIVACAT DNA was deposited on Jan. 13, 2012, underaccession number DSM 25555 at the DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen, Inhoffenstraβe 7 B, D-38124Braunschweig, Federal Republic of Germany.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the gene included in the expressionplasmid. Dots identify start and stop codons.

FIG. 2. Cellular immune responses analyzed in pooled splenocytes by flowcytometric analysis. Frequency of total Gag, Pol, and Nef-Tat-Vifspecific interferon gamma responses among groups in FIG. 2A anddistribution of CD4 and CD8 responses in FIG. 2B are shown.

FIG. 3. Responses to Gag, Pol, NTV and the HIVACAT T cell immunogensequence measured by interferon gamma ELISpot assay in murinesplenocytes. The individual mice were immunized with the plasmidsencoding for the full Gag, Pol and Nef-Tat-Vif polypeptide. Contributionof the responses targeting the regions included in the HIVACAT T cellimmunogen to the total interferon gamma Gag-Pol-NTV specific response isshown.

FIG. 4. Comparison of the breadth in FIG. 4A and magnitude in FIG. 4B ofthe interferon gamma responses targeting the HIVACAT T cell immunogen inimmunized mice. The subjects were treated with either the plasmidsencoding the full proteins or the minimal T cell sequence.

FIG. 5. Balance of interferon gamma responses against Gag, Pol, Vif orNef for mice immunized with 20 μg of plasmids encoding full Gag, Polplus Nef-Tat-Vif polypeptide and HIVACAT T cell immunogen. Dominance ofGag-specific responses is shown in FIG. 5A for mice immunized with fullproteins whereas a more balanced repertoire is seen in FIG. 5B for miceimmunized with the HIVACAT T cell immunogen.

FIG. 6. Binding antibodies to p24, p37 and p55 detected by Westernimmunoblot by using cell extracts from HEK 293 cells transfected withthe 1 mg of gag and gag-pol expression vectors (showing p55 gag, andprocessed p24, p37 and p55 gag subunits) separated on 12% SDS-Page andprobing the membranes with a) human sera of and HIV-infected patient(FIG. 6A), b) pooled sera from mice immunized with high doses of theimmunogen (FIG. 6B) and c) pooled sera from mice immunized with lowdoses of the immunogen (all at a 1:100 dilution) (FIG. 6C).

FIG. 7. a) Endpoint titers of Gag-p24 specific binding antibody fromtreated mice (FIG. 7A). The determination was performed by ELISA fromindividual serial 4-fold diluted pooled serum samples. b) In housedeveloped gag p55 ELISA using the HIV-1IIIB pr55 Gag recombinant protein(Cat. No. 3276, NIH Reagent Program, Bethesda, Md., US) (FIG. 7B). Thedetermination was performed in individual mice sera at 1:100 dilution.

FIG. 8. a) Schematic representation of mice immunizations (FIG. 8A).Groups of six C57BL/6 mice were used to compare immunogenicity of thedifferent heterologous combinations (2×DNA prime vs 3×DNA prime followedby 1×MVA boost) using either 100 μg of pDNA-HIVACAT or 10{circumflexover ( )}6 pfu of MVA-HIVACAT by intramuscular injection. b) Comparisonof the breadth and magnitude of the IFNγ responses targeting HIVACAT Tcell immunogen in individual immunized mice (FIG. 8B). c) Distributionof Gag, Pol, Vif and Nef specific responses in individual immunized mice(FIG. 8C). d) Distribution among the 8 protein subunits included in theHIVACAT T cell immunogen (Gag p17, Gag p24, Gag p2p7p1p6, Pol-Protease,Pol-RT, Pol-Integrase, Vif and Nef) in different immunization groups isshown (FIG. 8D).

DETAILED DESCRIPTION OF THE INVENTION

The invention discloses several immunogenic compounds effective forinducing a high immune response against HIV in a broad range ofsubjects. In particular, HIV-specific CD4⁺ and CD8⁺ T cell responses tokey HIV-encoded epitopes have been obtained with these compounds.

1. Definitions of General Terms and Expressions

The term “adjuvant”, as used herein, refers to an immunological agentthat modifies the effect of an immunogen, while having few if any directeffects when administered by itself. It is often included in vaccines toenhance the recipient's immune response to a supplied antigen, whilekeeping the injected foreign material to a minimum. Adjuvants are addedto vaccines to stimulate the immune system's response to the targetantigen, but do not in themselves confer immunity. Non-limiting examplesof useful adjuvants include mineral salts, polynucleotides,polyarginines, ISCOMs, saponins, monophosphoryl lipid A, imiquimod,CCR-5 inhibitors, toxins, polyphosphazenes, cytokines, immunoregulatoryproteins, immunostimulatory fusion proteins, co-stimulatory molecules,and combinations thereof. Mineral salts include, but are not limited to,AIK(SO₄)₂, AlNa(SO₄)₂, AlNH(SO₄)₂, silica, alum, Al(OH)₃, Ca₃(PO₄)₂,kaolin, or carbon. Useful immunostimulatory polynucleotides include, butare not limited to, CpG oligonucleotides with or without immunestimulating complexes (ISCOMs), CpG oligonucleotides with or withoutpolyarginine, poly IC or poly AU acids. Toxins include cholera toxin.Saponins include, but are not limited to, QS21, QS17 or QS7. An exampleof a useful immunostimulatory fusion protein is the fusion protein ofIL-2 with the Fc fragment of immunoglobulin. Useful immunoregulatorymolecules include, but are not limited to, CD40L and CD1a ligand.Cytokines useful as adjuvants include, but are not limited to, IL-1,IL-2, IL-4, GMCSF, IL-12, IL-15, IGF-1, IFNα, IFN-β, and interferongamma. Also, examples of are muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), RIBI (MPL+ TDM+CWS) in a 2percent squalene/TWEEN® 80 emulsion, lipopolysaccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g.poly IC and poly AU acids), wax D from Mycobacterium tuberculosis,substances found in Corynebacterium parvum, Bordetella pertussis, andmembers of the genus Brucella, Titermax, Quil A, ALUN, Lipid Aderivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives,synthetic peptide matrixes or GMDP, Montanide ISA-51 and QS-21, CpGoligonucleotide, poly I:C, and GMCSF. See Osol A., Ed., Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa., US, 1980, pp.1324-1341), Hunter R, U.S. Pat. No. 5,554,372, and Jager E, Knuth A,WO1997028816. Combinations of adjuvants can also be used.

The term “AIDS”, as used herein, refers to the symptomatic phase of HIVinfection, and includes both Acquired Immune Deficiency Syndrome(commonly known as AIDS) and “ARC,” or AIDS-Related Complex. See AdlerM, et al., Brit. Med. J. 1987; 294: 1145-1147. The immunological andclinical manifestations of AIDS are well known in the art and include,for example, opportunistic infections and cancers resulting from immunedeficiency.

The term “amino acid linker”, as used herein, refers to an amino acidsequence other than that appearing at a particular position in thenatural protein and is generally designed to be flexible or to interposea structure, such as an α-helix, between two protein moieties. A linkeris also referred to as a spacer. The linker is typically non-antigenicand can be of essentially any length (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids). The linkermay also be a location or sequence where the cellular antigen processingmachinery can initiate the degradation of the immunogenic polypeptidewithout destroying potent T cell epitopes).

The term “antiretroviral resistance mutation site”, as used herein,relates to a site that, when mutated, confers resistance to anantiretroviral agent. Such sites can be identified by mining knowndatabases such as the Stanford University HIV Drug Resistance Database,where, for example, sequences and treatments from viruses with specificmutations or drug susceptibility data for isolates with selectedmutations can be retrieved. Assays for testing drug resistance of HIVare known in the art. See Dong J, US 20040106136 and Shafer R, Assay forAntiretroviral Resistance, HIV InSite Knowledge Base Chapter (January2012). Already known antiretroviral resistance mutation sites in HIV areregularly published by the World Health Organization or the by theInternational Antiviral Society-USA (e.g. Johnson V, et al., ISA-USATopics Antiviral Med. 2011; 19(4): 153-164.

The expression “cellular immune response”, as used herein, describes animmune response against foreign antigen(s) that is mediated by T cellsand their secretion products.

The term “center-of-tree sequence” or “COT”, as used herein, refers to asequence from which the average evolutionary distance to each tip of aphylogenetic diagram of related variant sequences has been minimized.See Nickle D, et al., Science 2003; 299, 1515-1517.

The term “codon optimized”, as used herein, relates to the alteration ofcodons in nucleic acid molecules to reflect the typical codon usage ofthe host organism without altering the polypeptide encoded by the DNA,to improve expression. A plethora of methods and software tools forcodon optimization have been reported previously. See Narum D, et al.,Infect. Immun. 2001; 69(12):7250-7253, Outchkourov N, et al., ProteinExpr. Purif 2002; 24(1):18-24, Feng L, et al., Biochemistry 2000;39(50):15399-15409, and Humphreys D, et al., Protein Expr. Purif. 2000;20(2):252-2.

The term “comprising” or “comprises”, as used herein, discloses also“consisting of” according to the generally accepted patent practice.

The expression “disease associated with a HIV infection”, as usedherein, includes a state in which the subject has developed AIDS, butalso includes a state in which the subject infected with HIV has notshown any sign or symptom of the disease. Thus, the vaccine of theinvention when administered to a subject that has no clinical signs ofthe infection can have a preventive activity, since they can prevent theonset of the disease. The immunogenic compositions are capable ofpreventing or slowing the infection and destruction of healthy CD4+ Tcells in such a subject. It also refers to the prevention and slowingthe onset of symptoms of the acquired immunodeficiency disease such asextreme low CD4+ T cell count and repeated infections by opportunisticpathogens such as Mycobacteria spp Pneumocystis carinii, andPneumocystis cryptococcus. Beneficial or desired clinical resultsinclude, but are not limited to, an increase in absolute naive CD4+ Tcell count (range 10-3520), an increase in the percentage of CD4+ T cellover total circulating immune cells (range 1-50 percent), and/or anincrease in CD4+ T cell count as a percentage of normal CD4+ T cellcount in an uninfected subject (range 1-161 percent).

The terms “variant” or “fragment”, as used herein, refer to apolypeptide derived from any of SEQ ID NOs 1-16 by deletion of one ormore terminal amino acids at the N-terminus or at the C-terminus of anindividual SEQ ID NO. Variant or fragments preferably have a length ofat least 8 amino acids or up to 10%, up to 20%, up to 30%, up to 40%, upto 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 99% of itsrespective SEQ ID NO.

The term “HIV genome”, as used herein, refers to a RNA sequenceapproximately 8749 nucleotide long enclosed by the HIV capsid andencoding the genes gag, pol, env, tat, rev, vif, nef, vpr, vpu, vpx, andoptionally, tev. The HIV genome sequence underlies high variability, forthis reason, the HIV genome referred to in the invention is not limitedto any specific sequence. Preferred sequences are those of the HIV typesand subtypes recited herein.

The term “human immunodeficiency virus” or “HIV”, as used herein, referhuman immunodeficiency viruses generically and includes HIV type 1(“HIV-1”), HIV type 2 (“HIV-2”) or other HIV viruses, including, forexample, the HIV-1, HIV-2, emerging HIV and other HIV subtypes and HIVvariants, such as widely dispersed or geographically isolated variantsand simian immunodeficiency virus (“SIV”). For example, an ancestralviral gene sequence can be determined for the env and gag genes ofHIV-1, such as for HIV-1 subtypes A, B, C, D, E, F, G, H, J, and K, andintersubtype recombinants such as AG, AGI, and for groups M, N, O or forHIV-2 viruses or HIV-2 subtypes A or B. HIV-1, HIV-2 and SIV include,but are not limited to, extracellular virus particles and the forms ofthe viruses associated with their respective infected cells.

The “humoral immune response”, as used herein, describes an immuneresponse against foreign antigen(s) that is mediated by antibodiesproduced by B cells.

The term “immunogenic composition”, as used herein, refers to acomposition that elicits an immune response that produces antibodies orcell-mediated immune responses against a specific immunogen.

The term “immunogenic polypeptide” or “immunogen”, as used herein,refers to a polypeptide antigen that is able to induce an adaptiveimmune response (i.e. a humoral or cell-mediated immune response), ifinjected on its own or with an adjuvant.

The term “kit”, as used herein, refers to a combination of articles thatfacilitate a process, method or application. These kits provide thematerials necessary for carrying out the application described in thepresent invention.

The term “operably linked”, as used herein, is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). See Auer H, NatureBiotechnol. 2006; 24: 41-43.

The term “peptide tag” or “tag”, as use herein, refers to a peptide oramino acid sequence, which can be used in the isolation or purificationof said immunogen. Thus, said tag is capable of binding to one or moreligands, for example, one or more ligands of an affinity matrix such asa chromatography support or bead with high affinity. Illustrative,non-limitative, examples of tags useful for isolating or purifying aprotein include Arg-tag, FLAG-tag, His-tag, or Strep-tag; an epitopecapable of being recognized by an antibody, such as c-myc-tag(recognized by an anti-c-myc antibody), SBP-tag, S-tag, calmodulinbinding peptide, cellulose binding domain, chitin binding domain,glutathione S-transferase-tag, maltose binding protein, NusA, TrxA, DsbAor Avi-tag; an amino acid sequence, such as AHGHRP (SEQ ID NO:53),PIHDHDHPHLVIHS (SEQ ID NO:54), or GMTCXXC (SEQ ID NO:55); orβ-galactosidase. See Terpe K, et al., Appl. Microbiol. Biotechnol. 2003;60:523-525.

The terms “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable diluent,” or “pharmaceutically acceptable excipient”, or“pharmaceutically acceptable vehicle,” as used interchangeably herein,refer to a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any conventionaltype. A pharmaceutically acceptable carrier is essentially non-toxic torecipients at the employed dosages and concentrations and is compatiblewith other ingredients of the formulation. For example, the carrier fora formulation containing polypeptides would not include normallyoxidizing agents and other compounds known to be deleterious topolypeptides. Suitable carriers include, but are not limited to, water,dextrose, glycerol, saline, ethanol, and combinations thereof. Thecarrier can contain additional agents such as wetting or emulsifyingagents, pH buffering agents, or adjuvants that enhance the effectivenessof the formulation.

The terms “prevent,” “preventing,” and “prevention”, as used herein,refer to inhibiting the inception or decreasing the occurrence of adisease in an animal. Prevention may be complete (e.g. the total absenceof pathological cells in a subject). The prevention may also be partial,such that for example the occurrence of pathological cells in a subjectis less than that which would have occurred without the presentinvention. Prevention also refers to reduced susceptibility to aclinical condition.

The term “secretion signal peptide” refers to a highly hydrophobic aminoacid sequence (e.g. preferably 15 to 60 amino acids long) of proteinsthat must cross through membranes to arrive at their functioningcellular location. By binding to signal recognition particles, thesesequences direct nascent protein-ribosome complexes to a membrane wherethe protein is inserted during translation. Signal peptides directtranslational uptake of the protein by various membranes (e.g.endoplasmic reticulum, mitochondria, chloroplast, peroxisome). Leadersignal sequences on non-membrane proteins are ultimately removed byspecific peptidases. Some signal peptides used include MCP-3 chemokine,for promoting secretion and attraction of antigen presenting cells; acatenin (CATE)-derived peptide for increased proteasomal degradation;and the lysosomal associated protein, LAMP1 for targeting the MHC IIcompartment. See Rosati M, et al., Proc. Natl. Acad. Sci. USA 2009;106:15831-15836.

The expression “sequential administration”, as used herein, means thatthe administration is not simultaneous, but a first administration isperformed, followed by one or more successive administrations.

The expression “substantially preserves the immunological capabilitiesof the immunogenic polypeptide”, as used herein, means that the variantshows at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or 100% of the ability of the immunogenic polypeptide for inducingan adaptive immune response (i.e. a humoral or cell-mediated immuneresponse), if injected on its own or with adjuvants.

The term “treat” or “treatment”, as used herein, refers to theadministration of an immunogenic composition of the invention or of amedicament containing it to control the progression of the diseasebefore or after clinical signs have appeared. Control of the diseaseprogression is understood to mean the beneficial or desired clinicalresults that include, but are not limited to, reduction of the symptoms,reduction of the duration of the disease, stabilization of pathologicalstates (specifically to avoid additional deterioration), delaying theprogression of the disease, improving the pathological state andremission (both partial and total). The control of progression of thedisease also involves an extension of survival, compared with theexpected survival if treatment was not applied.

The term “vaccine”, as used herein, refers to a substance or compositionthat establishes or improves immunity to a particular disease byinducing an adaptive immune response including an immunological memory.A vaccine typically contains an agent that resembles a disease-causingmicroorganism or a part thereof (e.g. a polypeptide). Vaccines can beprophylactic or therapeutic.

The term “variant”, as used herein, refers to all those amino acidsequences derived from any of SEQ ID NOs 1-16 by means of modificationsor mutations, including substitutions, preferably conservativesubstitutions, insertions or non-terminal deletions, affecting one ormore amino acids and which substantially preserves the immunogeniccapabilities of the immunogenic polypeptide.

The term “vector”, as used herein, refers to a nucleic acid molecule,linear or circular, that comprises a segment according to the nucleicacid of interest operably linked to additional segments that provide forits autonomous replication in a host cell of interest or according tothe expression cassette of interest.

2. Immunogenic Polypeptides of the Invention

In a first aspect, the invention relates to an immunogenic polypeptidehaving an amino acid sequence comprising the sequences SEQ ID NOs 1-16or variants of said SEQ ID NO:1-16, wherein each of said variants has alength of at least 8 amino acids, with the provisos that said amino acidsequence does not comprise any sequence stretches derived from the HIVgenome of a length of 8 or more amino acids other than an amino acidsequence according to any of SEQ ID NOs 1-16 or the variants thereof.

In a particular embodiment, the immunogenic polypeptide of the firstaspect has an amino acid sequence comprising SEQ ID NO: 49.

In a second aspect, the invention relates to an immunogenic polypeptidehaving an amino acid sequence comprising at least one sequence selectedfrom the group consisting of the SEQ ID NOs 1-16 or variants thereof ora fragment thereof, wherein said fragment has a length of at least 8amino acids, with the provisos that:

i) said amino acid sequence does not comprise any sequence stretchesderived from the HIV genome of a length of 8 or more amino acids otherthan an amino acid sequence according to any of SEQ ID NOs 1-16 or avariant or a fragment thereof, and

ii) when the immunogen comprises only one sequence selected from thegroup consisting of SEQ ID NOs 1-16, then this sequence is not selectedfrom the group consisting of SEQ ID NO: 3, 5, 6 and 16.

Preferably, the variant according to the first and second aspects isequivalent to its related sequence and derives from a different HIVstrain or is an artificial HIV sequence. Equivalent in this respectmeans different in one or more amino acid residues, but corresponding tothe same sequence (e.g. determined by the position in the genome orsequence similarity). In other words, in a preferred embodiment, thevariant is a “naturally occurring variant”, which refers to nucleic acidsequences derived from an HIV genome of a presently or formerlycirculating virus and can be identified from existing databases (e.g.GenBank and Los Alamos sequence databases). The sequence of circulatingviruses can also be determined by molecular biology methodologies. SeeBrown T, “Gene Cloning” (Chapman & Hall, London, G B, 1995); Watson R,et al., “Recombinant DNA”, 2nd Ed. (Scientific American Books, New York,N.Y., US, 1992); Sambrook J, et al., “Molecular Cloning. A LaboratoryManual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,US, 1989). Preferably, a variant of any of SEQ ID NOs 1-16 has an aminoacid sequence identity of at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or at least 99% to its corresponding (i.e., SEQID NOs 1-16). Examples of algorithms suitable for determining percentsequence identity and sequence similarity are BLAST and BLAST 2.0algorithms. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402and Altschul S, et al., J. Mol. Biol. 1990; 215:403-410. The BLAST andBLAST 2.0 programs can be used to determine percent sequence identityfor the nucleic acids and proteins of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information.

Variants may also contain one or more modified amino acid residues (e.g.residues that are modified by the attachment of substituent groups), orone or more unnatural amino acids such as beta amino acids.

Methods for determining the extent of the cellular response are known inthe art. Any method suitable for assessing the stimulation of T cells inresponse to an Ag can be used. The procedures described below provide afew examples of suitable methods:

1) Enzyme-linked immunospot (ELISpot): non-adherent cells frompre-culture wells are transferred to a plate, which has been coated withthe desired anti-cytokine capture antibodies (Abs; e.g. anti-IFN,-IL-10, -IL-2, -IL-4). Revelation is carried out with biotinylatedsecondary Abs and standard colorimetric or fluorimetric detectionmethods such as streptavidin-alkaline phosphatase and NBT-BCIP and thespots counted. ELISpot readouts are then expressed as spot-forming cells(SFC)/10⁶ input cells.

2) Supernatant cytokine assay: cytokines released in the culturesupernatant are measured by different techniques, such as enzyme-linkedimmunosorbent assays (ELISA), BD cytometric bead array, Biorad Bio-Plexassay and others.

3) HLA Class I tetramers: with this procedure, Ag-reactive T cellsrecognizing specific peptide epitopes are detected, using eithercommercially available reagents (e.g. MEW Class I Dexamers, Immudex,Copenhagen, DK) or in-house generated ones (e.g. Novak E, et al., J.Clin. Invest. 1999; 104:R63-R67).

4) HLA Class II tetramers: with this procedure, Ag-reactive T cellsrecognizing specific peptide epitopes are detected, using eithercommercially available reagents (e.g. MEW Class II Ultimers™, ProImmuneLtd, Oxford, GB) or in-house generated ones (e.g. Novak, 1991, supra).

5) Upregulation of activation markers (e.g. CD69, CD25, CD137): withthis procedure, Ag-specific T cell responses are detected by theirdifferential expression of activation markers exposed on the membranefollowing Ag-recognition.

6) Cytokine capture assays: this system is a valid alternative to theELISpot to visualize Ag-specific T cells according to their cytokineresponse (Miltenyi Biotec GmbH, Bergisch Gladbach, DE). In addition, itallows the direct sorting and cloning of the T cells of interest.

7) CD154 assay: this procedure is limited to detection of Ag-specificCD4+ T cells. See Chattopadhyay P, et al., Nat. Med. 2005; 11:1113-11117and Frentsch M, et al., Nat. Med. 2005; 11:1118-1124.

8) CD107 assay: this procedure allows the visualization of Ag-specificCD8+ T cells with cytotoxic potential. See Betts M, et al., J. Immunol.Methods 2003; 281:65-78.

9) CFSE dilution assay: this procedure detects Ag-specific T cells (CD4+and CD8+) according to their proliferation following Ag recognition. SeeMannering S, et al., J. Immunol. Methods 2003; 283:173-183.

Methods for determining the extent of the humoral response of a variantare known in the art. Any method suitable for assessing the stimulationof T cells in response to an Ag can be used. Examples of suitablemethods include, but are not limited to, detecting or quantitating therelative amount of an antibody, which specifically recognizes anantigenic or immunogenic agent in the sera of a subject who has beentreated with an immunogenic polypeptide or variant relative to theamount of the antibody in an untreated subject. Antibody titers can bedetermined using standard assays such as enzyme-linked immunosorbentassay (ELISA), Single Radial Immunodiffussion Assay (SRID), or EnzymeImmunoassay (ETA).

In a preferred embodiment, the variant of any of SEQ ID NOs 1-16 is afragment of said sequence(s).

In specific embodiments, ancestral viral sequences are determined forthe env genes of HIV-1 subtypes B or C, or for the gag genes of subtypesB or C. In other embodiments, the ancestral viral sequence is determinedfor other HIV genes or polypeptides, such as pol or the auxiliary genesor polypeptides. In yet another embodiment, the viral sequence isdetermined by consensus or center-of-tree techniques.

In a preferred embodiment, the HIV is a group M HIV. Group M is thepredominant circulating HIV-1 group. It has been divided into subtypes,denoted with letters, and sub-subtypes, denoted with numerals. SubtypesA1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J, and K are currentlyrecognized. HIV-1 subtypes, also called clades, are phylogeneticallylinked strains of HIV-1 that are approximately the same genetic distancefrom one another; in some cases, subtypes are also linked geographicallyor epidemiologically. Genetic variation within a subtype can be 15 to 20percent or more, whereas variation between subtypes or divergent membersof the same subtype is usually 25 to 35 percent. Advances in full-genomesequencing of HIV have led to the identification of circulating andunique recombinant forms (CRFs and URFs, respectively). These are theresult of recombination between subtypes within a dually infectedperson, from whom the recombinant forms are then passed to other people.The recombinant progeny are classified as circulating recombinant formsif they are identified in three or more people with no directepidemiologic linkage; otherwise they are described as uniquerecombinant forms.

In one embodiment, said immunogen has an amino acid sequence comprisingat least one sequence selected from the group consisting of the SEQ IDNOs 1-16 or variants thereof, wherein said proviso ii) is: when theimmunogen comprises only one sequence selected from the group consistingof SEQ ID NOs 1-16, then this sequence is not selected from the groupconsisting of SEQ ID NOs 1-16.

In a preferred embodiment, said immunogen comprises at least two, atleast three, or at least four sequences selected from the groupconsisting of the SEQ ID NOs 1-16 or variants thereof, wherein saidproviso ii) is: when the immunogen comprises only two, three, or foursequences selected from the group consisting of SEQ ID NOs 1-16, thennot all of these sequences are selected from the group consisting of SEQID NO: 3, 5, 6 and 16. In another embodiment, said immunogen has anamino acid sequence comprising at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten sequences selected from the group consistingof the SEQ ID NOs 1-16 or variants thereof, wherein said proviso ii) is:when the immunogen comprises only two, three, four, five, six, seven,eight, nine or ten sequences selected from the group consisting of SEQID NOs 1-16, then not all of these sequences are selected from the groupconsisting of SEQ ID NOs 1-16.

In a preferred embodiment, the immunogen according to the first aspectcomprises the sequences according to SEQ ID NOs 1-16 or variants thereofin the order 1-16.

In one embodiment, the invention relates to the immunogen of the firstaspect wherein at least two sequences are adjoined by an amino acidlinker.

In another embodiment, the invention relates to the immunogen of thesecond aspect, wherein, if said immunogen comprises at least twosequences selected from the group consisting of the SEQ ID NOs 1-16 orvariants thereof, said sequences are adjoined by an amino acid linker.

In a preferred embodiment of the immunogens of both the first and secondaspect, the linker has the amino acid sequence A, AA or AAA. In aanother embodiment, when the C-terminal residue of the sequence locatedN-terminally with respect to the linker or the N-terminal residue of thesequence located C-terminally is an alanine residue, the linker can beshortened so that an AAA sequence is formed in the junction regionbetween adjoining sequences. Thus, in a preferred embodiment, if theC-terminal residue of the sequence located N-terminally with respect tothe linker is an alanine or if the N-terminal residue of the sequencelocated C-terminally with respect to the linker is alanine, the linkerhas the sequence AA. In another embodiment, if the C-terminal residue ofthe sequence located N-terminally with respect to the linker and theN-terminal residue of the sequence located C-terminally with respect tothe linker are both alanine, then the linker as the sequence A.

In another embodiment, said immunogens further comprise a secretionsignal peptide at the N-terminus, wherein said signal peptide preferablyenhances secretion of the immunogen from cells expressing saidimmunogen. A preferred secretion signal peptide is derived from GMCSF(granulocyte macrophage colony-stimulating factor), preferably followedby a valine to increase stability. The sequence of the GMCSF signalpeptide is preferably: MWLQSLLLLGTVACSIS (SEQ ID NO: 46) orMWLQSLLLLGTVACSISV (SEQ ID NO: 47).

In another embodiment, said immunogens further comprise optionally apeptide tag. The peptide tag can be located at the N-terminus betweenthe signal peptide and the immunogenic polypeptide or, preferably, canbe located at the C-terminus before the stop codon.

Preferably, said tag is a FLAG peptide. The FLAG system utilizes ashort, hydrophilic 8-amino acid peptide, which is fused to therecombinant protein of interest. The FLAG peptide includes the bindingsite for several highly specific ANTI-FLAG monoclonal antibodies (M1,M2, M5; Sigma-Aldrich Corp., Saint Louis, Mo., US), which can be used toassess expression of the protein of interest on material fromtransfected cells. Because of the small size of the FLAG peptide tag, itdoes not shield other epitopes, domains, or alter the function,secretion, or transport of the fusion protein generally. Preferably,said FLAG peptide has the sequence DYKDDDDKL (SEQ ID NO: 48).

In a preferred embodiment, said tag is only for expression analysis andpurification of the immunogen and it is removed before using it toelicit an immune response.

In another embodiment, the invention relates to said immunogens, whereinsaid amino acid sequence comprises at least one antiretroviralresistance mutation site.

The mutation can occur at any site within the viral genome. Preferably,the mutation occurs in the region encoding the integrase, the proteaseor the reverse transcriptase genes.

Mutants within the integrase that confer resistance to integraseinhibitors include, without limitation, T66, E92, F121, E138, G140,Y143, S147, Q148, S153, N155, E157 and R263 within SEQ ID NO: 1 and acombination thereof. In a preferred embodiment, the mutation is selectedfrom the group consisting of mutations E92Q, G140S, G 140A and Y143R inthe integrase protein and their combinations.

Mutants within the protease associated with protease inhibitor (PI)resistance include major protease, accessory protease, and proteasecleavage site mutations. See Shafer R, et al., AIDS Rev. 2008;10(2):67-84. Seventeen largely non-polymorphic positions are of the mostclinical significance, including L231, L241, D30N, V321, L33F, M461/L,147/V/A, G48V/M, 150L/V, F53L, 154V/T/A/L/M, G73S/T, L76V, V82A/T/F/S,184V/A/C, N88D/S, L90M. Accessory protease mutations include thepolymorphic mutations L101/V, 113V, K20R/M/I, M361, D60E, 162V, L63P,A71V/T, V771, and 193L and the non-polymorphic mutations L10F/R, V111,E34Q, E35G, K43T, K451, K55R, Q58E, A711/L, T74P/A/S, V751, N83D,P79A/S, 185V, L89V, T91S, Q92K and C95F.

In another embodiment, the antiretroviral resistance mutation site islocated in the reverse transcriptase, resulting in a resistance tonucleoside reverse transcriptase inhibitor (NRTI) or to non-nucleosidereverse transcriptase inhibitor (NNRTI). The NRTI resistance mutationsinclude M184V, thymidine analog mutations (TAMs), mutations selected byregimens lacking thymidine analogs (Non-TAMs), and multi-nucleosideresistance mutations (Multi-NRTI mutations) and many recently describednon-polymorphic accessory mutations. Altogether, M184V,non-thymidine-analog-associated mutations such as K65R and L74V, and themulti-nucleoside resistance mutation Q151M act by decreasing NRTIincorporation. Thymidine analog mutations, the T69 insertions associatedwith multi-nucleoside resistance, and many of the accessory mutationsfacilitate primer unblocking. See Shafer, 20008, supra. M184V is themost commonly occurring NRTI resistance mutation. The most commondrug-resistant amino acid mutations are M41L, D67N, K70R, L210W, T215Y/Fand K219QE. The most common mutations in patients developing virologicfailure while receiving a non-thymidine analog containing NRTI backbone(Non-TAMs) include M184V alone or M184V in combination with K65R orL74V. Other Non-TAMs mutations include K65N, K70E/G, L741, V75T/M,Y115F. Amino acid insertions at codon 69 generally occur in the presenceof multiple TAM, and in this setting are associated with intermediateresistance to 3TC and FTC and high-level resistance to each of theremaining NRTI. Q151M is a 2-bp mutation (CAG.fwdarw.ATG) that isusually accompanied by two or more of the following mutations: A62V,V751, F77L, and F116Y. The Q151M complex causes high-level resistance toZDV, d4T, ddI, and ABC, and intermediate resistance to TDF, 3TC, andFTC. See Shafer R, et al., AIDS Rev. 2008; 10(2):67-84

NNTRI resistance mutations include, without limitation, the primaryNNRTI resistance mutations (K103N/S, V106A/M, Y181C/I/V, Y188L/C/H, andG190A/S/E), the NNRTI resistance secondary mutations (L1001, K101P,P225H, F227L, M230L, and K238T) and rate mutations (V179F, F227C andL2341).

Minor non-polymorphic mutations—A98G, K101 E, V 1081, and V 179D/E arecommon NNRTI resistance mutations that reduce susceptibility tonevirapine and efavirenz about 2-fold to 5-fold.

Miscellaneous nonnucleoside reverse transcriptase inhibitor resistancemutations, such as K101Q, 1135T/M, V1791, and L2831, reducesusceptibility to nevirapine and efavirenz by about twofold and may actsynergistically with primary NNRTI resistance mutations. Other mutationssuch as L74V, H221Y, K223E/Q, L228H/R, and N3481 are selected primarilyby NRTI, yet also cause subtle reductions in NNRTI susceptibility.

Preferably, said antiretroviral resistance mutation site is located inany of SEQ ID NOs 9-11. More preferably, said antiretroviral resistancemutation site is amino acid residue 8 of SEQ ID NO: 9, wherein the aminoacid Leu is substituted by the amino acid Met.

In another embodiment, the variant or fragment has a length of 8 to 40amino acids, more preferably a length of 11 to 27 amino acids.Preferably, said variant or fragment does not comprise an amino acidlinker adjoining any of SEQ ID NOs 1-16. Furthermore, it is preferredthat the C-terminal amino acid of said variant or fragment is neither G,P, E, D, Q, N, T, S, nor C, as these residues do not form a C terminalanchor for HLA class I restricted T cell epitopes generally.

In a most preferred embodiment, said variant or fragment is selectedfrom the group consisting of the peptides according to SEQ ID NOs 17-45.

Further, it is envisaged that said variant or fragment is combined withor fused to a heat shock protein. The present invention also relates toa fusion protein comprising said variant or fragment and a heat shockprotein. Preferred heat shock proteins are Hsp10, Hsp20, Hsp30, Hsp40,Hsp60, Hsp70, Hsp90, gp96, or Hsp100.

In another embodiment, the variant or fragment is a variant or fragmentaccording to the first and second aspects. Preferably, said variant orfragment does not comprise an amino acid linker adjoining any of SEQ IDNOs 1-16. Furthermore, it is preferred that the C-terminal amino acid ofsaid variant or fragment is neither G, P, E, D, Q, N, T, S, nor C, asthese residues do not form a C terminal anchor for HLA class Irestricted T cell epitopes generally.

In a most preferred embodiment, said variant or fragment is selectedfrom the group consisting of the peptides according to SEQ ID NOs 17-45.

Further, it is envisaged that said variant or fragment is combined withor fused to a heat shock protein. The present invention also relates toa fusion protein comprising said variant or fragment and a heat shockprotein. Preferred heat shock proteins are Hsp10, Hsp20, Hsp30, Hsp40,Hsp60, Hsp70, Hsp90, gp96, or Hsp100.

3. Nucleic Acids, Vectors, Viruses and Cells of the Invention

In a third aspect, the present invention relates to a nucleic acidencoding for the immunogen of the first aspect, and to an expressioncassette, a vector, a virus and a cell comprising said nucleic acid.

Preferably, said nucleic acid is a polynucleotide, referring tosingle-stranded or double-stranded polymers of nucleotide monomers(nucleic acids), including, but not limited to, 2′-deoxyribonucleotides(DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiesterbond linkages. The polynucleotides of the invention encode the immunogenof the invention without substantially comprising additional regions ofthe HIV genome.

In one embodiment, said nucleic acid is codon optimized. In a preferredembodiment, the nucleic acid is codon optimized for expression inhumans. Codon-optimized nucleic acids for use according to the presentinvention can be prepared by replacing the codons of the nucleic acidencoding the immunogen by “humanized” codons (i.e. the codons are thosethat appear frequently in highly expressed human genes). See André S, etal., J. Virol. 1998; 72:1497-1503. In a preferred embodiment, saidcodon-optimized nucleic acid has the sequence according to SEQ ID NO:50.

The nucleic acid of the third aspect may require cutting withrestriction enzymes in order to it ligate into a vector. This procedurecould entail the removal of various terminal nucleotides (e.g. 1, 2, or3). As such, in one embodiment, the invention relates to said nucleicacid, wherein it has been cut at each end with a restriction enzyme.

In another embodiment, the present invention relates to an expressioncassette comprising the nucleic acid of the third aspect, a promotersequence and a 3′-UTR and optionally a selection marker. Preferably, thepromoter sequence is a human cytomegalovirus (CMV) promoter or anearly-late p7.5 promotor sequence. Preferably, the 3′-UTR is a bovinegrowth hormone (BGH) poly-A. The optional selection marker is anantibiotic resistance gene (e.g. kanamycin, ampicilin, tetracycline,spectinomycin) preferably.

In yet another embodiment, the present invention relates to anexpression vector comprising the nucleic acid or the expression cassetteof the third aspect.

In one embodiment, the vector is an expression vector. Thus, suitablevectors according to the present invention include prokaryotic vectors,such as pUC18, pUC19, and Bluescript plasmids and derivatives thereof,like the mp18, mp19, pBR322, pMB9, ColE1, pCR1 and RP4 plasmids; phagesand shuttle vectors, such as pSA3 and pAT28 vectors; expression vectorsin yeasts, such as 2-micron plasmid type vectors; integration plasmids;YEP vectors; centromeric plasmids and analogues; expression vectors ininsect cells, such as the vectors of the pAC series and of the pVLseries; expression vectors in plants, such as vectors of the pIBI,pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series andanalogues; and expression vectors in superior eukaryotic cells eitherbased on viral vectors (e.g. MVA, adenoviruses, viruses associated toadenoviruses, retroviruses and lentiviruses) as well as non-viralvectors, such as the pSilencer 4.1-CMV (Ambion®, Life TechnologiesCorp., Carslbad, Calif., US), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1,pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6N5-His,pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1 vectors.

In a particular embodiment the expression vector is a mammalianexpression vector comprising a mammalian promoter and a polyadenylationsite. Preferably, the promoter is the human cytomegalovirus (CMV)promoter. Preferably, the polyadenylation site is the bovine growthhormone (BGH) polyadenylation site. The mammalian expression vector canbe modified to optimize vector replication in bacteria. The mammalianexpression vector can further comprise a selection gene, for example, agene coding a protein conferring resistance to an antibiotic. In aparticular embodiment, the mammalian expression vector comprises akanamycin resistance gene.

In other particular embodiment, the expression vector is a viral vector,preferably a Modified Vaccine Ankara (MVA) virus vector.

In another embodiment, the present invention relates to a viruscontaining the nucleic acid of the third aspect. Suitable viruses aresafe, have low toxicity and are genetically stable. Non-limitingexamples are retroviruses, particularly poxviruses such as MVA,lentiviruses, adenoviruses and adeno-associated viruses (AAVs).

In a further particular preferred embodiment the present inventionrelates to a recombinant Modified Vaccinia virus Ankara (MVA) comprisingin a polynucleotide or gene construct encoding the immunogenicpolypeptides of the invention. Modified Vaccinia Ankara (MVA) virus isrelated to the vaccinia virus, a member of the genera orthopoxvirus inthe family of poxyiridae. MVA has been generated by 516 serial passageson chicken embryo fibroblasts of the Ankara strain of vaccinia virus(CVA). See Mayr A, et al., Infection 1975; 3:6-14 and Sutter G, et al.,U.S. Pat. No. 6,440,422 and CH 568,392. MVA viruses are publiclyavailable (e.g. ATCC accession number VR-1508). MVA is distinguished byits attenuation (e.g. diminished virulence and limited ability toreproduce infectious virions in certain mammalian cells), whilemaintaining good immunogenicity and full capacity to replicate andproduce infectious virions in avian cells. Suitable MVA strains includestrains with enhanced safety due to i) capability of reproductivereplication in vitro in chicken embryo fibroblasts (CEF), but nocapability of reproductive replication in a human cell line, as in thehuman keratinocyte cell line HaCaT, the human embryo kidney cell line293, the human bone osteosarcoma cell line 143B, and the human cervixadenocarcinoma cell line HeLa; ii) failure to replicate in a mouse modelthat is incapable of producing mature B and T cells and as such isseverely immune compromised and highly susceptible to a replicatingvirus; and iii) induction of at least the same level of specific immuneresponse in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes. A suitableattenuated MVA strain in the strain referred to as MVA-BN. See ChaplinP, et al., WO2002042480, ECACC accession number V00083008.

In another embodiment, the present invention relates to a cellcomprising the nucleic acid, the expression cassette, the expressionvector, or the virus of the third aspect. Cells to be used can be of anycell type, including both eukaryotic cells and prokaryotic cells.Preferably, the cells include prokaryotic cells, yeast cells, ormammalian cells. Preferred examples of mammalian cells are COS cells,HeLa cells, HEK 293T cells or cells isolated from a patient (e.g. a HIVpatient).

4. Compositions of the Invention

In a fourth aspect, the present invention relates to a compositioncomprising a variant or fragment according to the first and secondaspects and a heat shock protein. Immunogenic compositions can beprepared, for instance, as injectables such as liquid solutions,suspensions, and emulsions. Preferred heat shock proteins are Hsp10,Hsp20, Hsp30, Hsp40, Hsp60, Hsp70, Hsp90, gp96, or Hsp100.

Furthermore, the invention relates to a pharmaceutical compositioncomprising an immunogen, nucleic acid, expression cassette, vector orcell according to the invention or a composition according to the fourthaspect and a pharmaceutically acceptable carrier. In one embodiment,said pharmaceutical compositions and the composition of the fourthaspect may be used as a vaccine, as laid out below.

5. Vaccine of the Invention

In another aspect, the present invention relates to a vaccine comprisingthe immunogen of the first and second aspects, the nucleic acid, theexpression cassette, the expression vector, the virus or the cell of thethird aspect or the composition of the fourth aspect.

In a preferred embodiment, said vaccine is capable of generatingcellular and humoral responses. More preferably, the vaccine generates acytotoxic T cell response. A cytotoxic T cell or cytotoxic T lymphocyte(CTL) assay can be used to monitor the cellular immune responsefollowing subgenomic immunization with a viral sequence againsthomologous and heterologous HIV strains. See Burke S, et al., J. Inf.Dis. 1994; 170:1110-1119 and Tigges M, et al., J. Immunol, 1996;156:3901-3910. Conventional assays utilized to detect T cell responsesinclude, for instance, proliferation assays, lymphokine secretionassays, direct cytotoxicity assays and limiting dilution assays. Forexample, antigen-presenting cells that have been incubated with apeptide can be assayed for their ability to induce CTL responses inresponder cell populations. Antigen-presenting cells can be cells suchas peripheral blood mononuclear cells (PBMCs) or dendritic cells (DCs).Alternatively, mutant non-human mammalian cell lines that are deficientin their ability to load MHC class I molecules with internally processedpeptides and that have been transfected with the appropriate human MHCclass I gene, can be used to test the capacity of a peptide of interestto induce in vitro primary CTL responses. PBMCs can be used as theresponder cell source of CTL precursors. The appropriateantigen-presenting cells are incubated with the peptide after which theprotein-loaded antigen-presenting cells are incubated with the respondercell population under optimized culture conditions. Positive CTLactivation can be determined by assaying the culture for the presence ofCTL that kill radiolabeled target cells, both specific peptide-pulsedtargets as well as target cells expressing endogenously processed formsof the antigen from which the peptide sequence was derived. For example,the target cells can be radiolabeled with ⁵¹Cr and cytotoxic activitycan be calculated from radioactivity released from the target cells.Another suitable method allows the direct quantification ofantigen-specific T cells by staining with fluorescein-labeled HLAtetrameric complexes. See Altman J, et al., Proc. Natl. Acad. Sci. USA1993; 90:10330-10334 and Altman J, et al., Science 1996; 274:94-96.Other relatively recent technical developments include staining forintracellular lymphokines and interferon release assays or ELISpotassays.

In one embodiment, the vaccine of the fourth aspect further comprisesone or more adjuvants or heat shock proteins.

Adjuvants are defines as above. Preferred heat shock proteins are Hsp10,Hsp20, Hsp30, Hsp40, Hsp60, Hsp70, Hsp90, gp96, or Hsp100.

6. Therapeutic Methods

In a preferred embodiment, the immunogenic polypeptide according to theinvention, the nucleic acid of the invention, the expression cassette ofthe invention, the expression vector of the invention, the virus of theinvention, the cell of the invention or the vaccine according to theinvention can be used in the prevention or treatment of an HIV infectionor a disease associated with an HIV infection.

Thus, in another aspect, the invention relates to the immunogenicpolypeptide according to the invention, the nucleic acid of theinvention, the expression cassette of the invention, the expressionvector of the invention, the virus of the invention, the cell of theinvention or the vaccine according to the invention for use in theprevention or treatment of an HIV infection or a disease associated withan HIV infection.

In another aspect, the invention relates to the use of the immunogenicpolypeptide according to the invention, the nucleic acid of theinvention, the expression cassette of the invention, the expressionvector of the invention, the virus of the invention, the cell of theinvention or the vaccine according to the invention for the manufactureof a medicament for the prevention or treatment of an HIV infection or adisease associated with an HIV infection.

In another aspect, the invention relates to a method for the preventionor treatment of an HIV infection or a disease associated with an HIV ina subject in need thereof comprising the administration to said subjectof the immunogenic polypeptide according to the invention, the nucleicacid of the invention, the expression cassette of the invention, theexpression vector of the invention, the virus of the invention, the cellof the invention or the vaccine according to the invention for themanufacture of a medicament for the prevention or treatment of an HIVinfection or a disease associated with an HIV infection.

In a particular embodiment, the immunogenic peptide, the nucleic acid,the expression cassette the expression vector, the virus, the cell orthe vaccine for use according to the invention, comprises the sequentialadministration of:

i) a first immunogenic peptide of any of claims 1-11, nucleic acid ofany of claims 12-14, expression cassette of claim 15, expression vectorof claim 16, virus of any of claims 17-18, cell of claim 19 or vaccineof claim 20 and

ii) a second immunogenic peptide of any of claims 1-11, nucleic acid ofany of claims 12-14, expression cassette of claim 15, expression vectorof claim 16, virus of any of claims 17-18, cell of claim 19 or vaccineof claim 20.

In a particular embodiment, the first the first immunogenic peptide,nucleic acid, expression cassette, expression vector, virus, cell orvaccine are different from the second immunogenic peptide, nucleic acid,expression cassette, expression vector, virus, cell or vaccine.Preferably, it is first administered an expression vector according tothe invention followed by the administration of a Modified VacciniaAnkara virus according to the invention.

In a particular embodiment, the first expression vector according to theinvention is administered at least twice, preferably at least threetimes.

The beneficial prophylactic or therapeutic effect of vaccine in relationto HIV infection or AIDS symptoms include, for example, preventing ordelaying initial infection of an individual exposed to HIV; reducingviral burden in an individual infected with HIV; prolonging theasymptomatic phase of HIV infection; maintaining low viral loads in HIVinfected patients whose virus levels have been lowered viaantiretroviral therapy (ART); increasing levels of CD4 T cells orlessening the decrease in CD4 T cells, both HIV-1 specific andnon-specific, in drug naive patients and in patients treated with ART,increasing the breadth, magnitude, avidity and functionality of HIVspecific CTL, increasing overall health or quality of life in anindividual with AIDS; and prolonging life expectancy of an individualwith AIDS. A clinician can compare the effect of immunization with thepatient's condition prior to treatment, or with the expected conditionof an untreated patient, to determine whether the treatment is effectivein inhibiting AIDS.

Preferably, said disease is AIDS, ARC or an HIV opportunistic disease.Non-limiting examples for HIV opportunistic diseases are Burkitt'slymphoma, candidiasis in the bronchi, trachea, lungs, or esophagus,cervical cancer, coccidioidomycosis (disseminated or outside the lungs),cryptococcosis (outside the lungs), cryptosporidiosis (in the intestineslasting for more than 1 month), cytomegalovirus infection (outside theliver, spleen, or lymph nodes), cytomegalovirus retinitis (with loss ofvision), HIV encephalopathy, herpes simplex lesions lasting for morethan one month, herpes simplex in the bronchi, lung, or esophagus,histoplasmosis (disseminated or outside the lungs), immunoblasticlymphoma, invasive cervical carcinoma (cancer), isosporiasis in theintestines lasting for more than one month, Kaposi's sarcoma, lymphoma(primary in the brain), Mycobacterium avium complex (disseminated oroutside the lungs), Mycobacterium kansasii (disseminated or outside thelungs), Mycobacterium tuberculosis (disseminated or outside the lungs),Pneumocystis carinii pneumonia, pneumonia (recurrent in 12 monthperiod), progressive multifocal leukoencephalopathy (PML), salmonellasepticemia (recurrent), toxoplasmosis (in the brain), wasting syndromeand any other disease resulting from an infection facilitated by acompromised immune system in an HIV-infected patient.

The vaccine of the invention may be useful for the therapy of HIV-1infection. While all animals that can be afflicted with HIV-1 or theirequivalents can be treated in this manner (e.g. chimpanzees, macaques,baboons or humans), the immunogenic compositions of the invention aredirected particularly to their therapeutic uses in humans. Often, morethan one administration may be required to bring about the desiredtherapeutic effect; the exact protocol (dosage and frequency) can beestablished by standard clinical procedures.

The present invention further relates to preventing or reducing symptomsassociated with HIV infection. These include symptoms associated withthe minor symptomatic phase of HIV infection, including, for example,shingles, skin rash and nail infections, mouth sores, recurrent nose andthroat infection and weight loss. In addition, further symptomsassociated with the major symptomatic phase of HIV infection include,for instance, oral and vaginal thrush (candidiasis), persistentdiarrhea, weight loss, persistent cough and reactivated tuberculosis orrecurrent herpes infections, such as cold sores (herpes simplex). Othersymptoms of full-blown AIDS which can be treated in accordance with thepresent invention include, for instance, diarrhea, nausea and vomiting,thrush and mouth sores, persistent, recurrent vaginal infections andcervical cancer, persistent generalized lymphadenopathy (PGL), severeskin infections, warts and ringworm, respiratory infections, pneumonia,especially Pneumocystis carinii pneumonia (PCP), herpes zoster (orshingles), nervous system problems, such as pains, numbness or “pins andneedles” in the hands and feet, neurological abnormalities, Kaposi'ssarcoma, lymphoma, tuberculosis or other similar opportunisticinfections.

Beneficial effects of the invention include, for example, preventing ordelaying initial infection of an individual exposed to HIV, reducingviral burden in an individual infected with HIV, prolonging theasymptomatic phase of HIV infection, maintaining low viral loads in HIVinfected patients whose virus levels have been lowered viaantiretroviral therapy (ART), increasing levels of CD4 T cells orlessening the decrease in CD4 T cells, both HIV-1 specific andnon-specific, in drug naïve patients and in patients treated with ART,increasing the breadth, magnitude, avidity and functionality of HIVspecific CTL, increasing overall health or quality of life in anindividual with AIDS and prolonging life expectancy of an individualwith AIDS. A clinician can compare the effect of immunization with thepatient's condition prior to treatment, or with the expected conditionof an untreated patient, or in a clinical trial of individuals treatedand untreated with the vaccine to determine whether the treatment iseffective in inhibiting AIDS.

The immunogenic compositions can be designed to introduce the nucleicacids or expression vectors to a desired site of action and release itat an appropriate and controllable rate. Methods of preparingcontrolled-release formulations are known in the art. For example,controlled release preparations can be produced by the use of polymersto complex or absorb the immunogen or immunogenic composition. Acontrolled-release formulation can be prepared using appropriatemacromolecules (e.g. polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) known to provide thedesired controlled release characteristics or release profile. Anotherpossible method to control the duration of action by acontrolled-release preparation is to incorporate the active ingredientsinto particles of a polymeric material (e.g. polyesters, polyaminoacids, hydrogels, polylactic acid, polyglycolic acid, copolymers ofthese acids, or ethylene vinylacetate copolymers). Alternatively,instead of incorporating these active ingredients into polymericparticles, it is possible to entrap these materials into microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (e.g. liposomes,albumin microspheres, microemulsions, nano-particles, nanocapsules) orin macroemulsions. See in Voller A, et al., Eds., “New Trends andDevelopments in Vaccines (University Park Press, Baltimore, Md., US,1978) and Gennaro A, Ed., “Remington's Pharmaceutical Sciences”, 18thEd. (Mack Publishing Co., Easton, Pa., US, 1990).

Suitable dosages of the nucleic acids and expression vectors of theinvention (collectively, the immunogens) in the immunogenic compositionof the invention can be readily determined by those of skill in the art.For example, the dosage of the immunogens can vary depending on theroute of administration and the size of the subject. Suitable doses canbe determined by those of skill in the art, for example by measuring theimmune response of a subject, such as a laboratory animal, usingconventional immunological techniques, and adjusting the dosages asappropriate. Such techniques for measuring the immune response of thesubject include but are not limited to, chromium release assays,tetramer binding assays, IFN ELISPOT assays, IL-2 ELISPOT assays,intracellular cytokine assays, and other immunological detection assays.See Harlow E, Lane D, “Antibodies: A Laboratory Manual” (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., US, 1988).

The immunogenic compositions can be administered using any suitabledelivery method including, but not limited to, intramuscular,intravenous, intradermal, transcutaneous, intranasal, mucosal (e.g.intrarectal, intravaginal, oral), and topical delivery. Such techniquesare well known in the art. More specific examples of delivery methodsare intramuscular injection, intradermal injection, and subcutaneousinjection. However, delivery need not be limited to injection methods.Further, delivery of DNA to animal tissue has been achieved by cationicliposomes direct injection of naked DNA into animal muscle tissue orintradermal injection of DNA using “gene gun” or electroporationtechnology. See Watanabe M, et al., Mol. Reprod. Dev. 1994; 38:268-274,Charnock-Jones D, et al., WO1996020013, Robinson H, et al., Vaccine1993: 11:957-960, Hoffman S, et al., Vaccine 1994; 12(16):1529-1533;Xiang Z, et al., Virology 1994; 199:132-140, Webster R, et al., Vaccine1994; 12:1495-1498, Davis H, et al., Vaccine 1994; 12: 1503-1509, DavisH, et al., Hum. Mol. Gen. 1993; 2:1847-1851, and Johnston S, et al.,Meth. Cell Biol. 1994; 43:353-365. Delivery can be accomplished via amucosal surface such as the anal, vaginal or oral mucosa also.

7. Kit of the Invention

In another aspect, the present invention relates to a kit comprising theimmunogen of the first aspect, the peptide or variant thereof of thesecond aspect, the nucleic acid, the expression cassette, the expressionvector, the virus or the cell of the fourth aspect, or the vaccine ofthe fourth aspect. These kits provide the materials necessary forcarrying out the application described in the present invention. The kitcould also be in the form of a patch.

In addition, the kit may comprise a packaging, which allows maintainingthe reagents within determined limits. Suitable materials for preparingsuch packings include glass, plastic (e.g. polyethylene, polypropylene,polycarbonate), bottles, vials, paper, or sachets. The kit of theinvention can additionally contain instructions for using the componentscontained therein, in particularly those constituting the hemostaticpatch of the invention. Said instructions can be found in the form ofprinted material or in the form of an electronic support which can storeinstructions such that they can be read by a subject, such as electronicstorage media (e.g. magnetic disks, tapes), or optical media (e.g.CD-ROM, DVD). The media can additionally or alternatively containinternet websites providing said instructions.

General Procedures

1. T Cell Immunogen Design

The following approach was followed for the design of the HIV OLPs ofthe invention.

Experimental (interferon gamma ELISpot) screening of 232 HIV infecteduntreated individuals using a consensus clade B peptide set revealedregions of the viral proteome that were predominantly targeted bysubjects with superior HIV control. See Frahm N, et al., J. Virol. 2004;78:2187-2200; Mothe B, et al., J. Transl. Med. 2011; 9(1):208. Theoverall test peptide set consisted of 410 18mer overlapping peptidesspanning the entire viral proteome. Of these, 26 OLPs were identifiedwhere the group of OLP responders had a significantly (p<0.05uncorrected for multiple comparison) reduced viral load compared to thegroup of OLP non-responders (i.e. individuals that did not react tothese OLPs in the interferon gamma ELISpot assay). These beneficial OLPshad a protective ratio (PR of >1) and were located in HIV Gag protein(n=10), in Pol (n=12), and in Vif (n=3) and Nef (n=1) proteins of thevirus. Of the 26 OLPs, 15 were partially overlapping. See Table 1.

Median Median viral viral load in load in Protective OLP ProteinProtein OLP clade B OLP re- OLP non- Ratio p- No. Protein sub-unitconsensus sequence sponders responders (PR)* value   3 Gag p17EKIRLRPGGKKKYKL  22947 39014 1.053 0.037 KHI   6 Gag p17 ASRELERFAVNPGLL 15380 43189 1.107 0.001   7 Gag p17 ERFAVNPGLLETSEGC  25939 38974 1.0400.049 R  10 Gag p17 QLQPSLQTGSEELRSL  16285 37237 1.085 0.031 Y  12 Gagp17 SLYNTVATLYCVHQR  23855 37113 1.044 0.037 IEV  23 Gag p24AFSPEVIPMFSALSEG  22947 37113 1.048 0.036 A  31 Gag p24 IAPGQMREPRGSDIA  3563 35483 1.281 0.028  34 Gag p24 STLQEQIGWMTNNPPI   6127 37360 1.2070.002 PV  48 Gag p24 ACQGVGGPGHKARV  12975 35755 1.107 0.041 LAEA  60Gag p15 GKIWPSHKGRPGNFL  16266 36434 1.083 0.044 QSR  75 Nef —WLEAQEEEEVGFPVR  13407 37360 1.108 0.026 PQV  76 Nef — EVGFPVRPQVPLRPM 59618 29855 0.937 0.001 TYK  84 Nef — NYTPGPGIRYPLTFGW  55402 305380.945 0.006 CF  85 Nef — RYPLTFGWCFKLVPV  69890 29903 0.924 0.002  90Nef — SLHGMDDPEKEVLV  89687 32650 0.911 0.042 WKF 159 Pol ProteaseKMIGGIGGFIKVRQYD  14736 36434 1.094 0.020 QI 160 Pol ProteaseFIKVRQYDQILIEICGH   3682 35755 1.277 0.031 K 161 Pol ProteaseQILIEICGHKAIGTVLV   9117 35483 1.149 0.050 163 Pol ProteaseLVGPTPVNIIGRNLLT  25965 45637 1.055 0.007 QI 171 Pol RT LVEICTEMEKEGKISK  1865 35483 1.391 0.014 I 181 Pol RT LDVGDAYFSVPLDKD  65858 32871 0.9370.041 FRK 195 Pol RT LRWGFTTPDKKHQKE   5624 37113 1.219 0.006 PPF 196Pol RT DKKHQKEPPFLWMG  10103 35483 1.136 0.044 YELH 210 Pol RTEIQKQGQGQWTYQIY  18155 35483 1.068 0.045 222 Pol RT PPLVKLWYQLEKEPIV412599 34640 0.808 0.030 GA 230 Pol RT IHLALQDSGLEVNIV  85102 341170.919 0.030 237 Pol RT VYLAWVPAHKGIGG  85102 34117 0.919 0.029 NEQV 240Pol RT SAGIRKVLFLDGIDKA 116902 32761 0.891 0.019 269 Pol IntegraseTKELQKQITKIQNFRV   6629 35755 1.192 0.030 YY 270 Pol IntegraseTKIQNFRVYYRDSRD  18171 37360 1.073 0.019 PLW 271 Pol IntegraseYYRDSRDPLWKGPAK  25939 35755 1.032 0.043 LLW 276 Pol IntegraseKIIRDYGKQMAGDDC   6629 35755 1.192 0.021 VA 279 Vpr — GPQREPYNEWTLELL 60222 32650 0.944 0.042 EEL 307 Env gp120 DLNNNTNTTSSSGEK 179419 341170.863 0.044 MEK 311 Env gp120 IRDKVQKEYALFYKL 179419 32871 0.860 0.008DVV 314 Env gp120 YRLISCNTSVITQACP  58206 31273 0.943 0.008 KV 315 Envgp120 SVITQACPKVSFEPIPI  61011 32871 0.944 0.034 H 320 Env gp120TNVSTVQCTHGIRPV 341587 34640 0.820 0.034 V 355 Env gp120 VAPTKAKRRVVQREK161602 34117 0.870 0.042 RAV 399 Env gp41 VIEVVQRACRAILHIP 388089 346400.812 0.026 RR 405 Vif — VKHHIMYISGKAKGW  16458 37237 1.084 0.021 FYRH406 Vif — GKAKGWFYRHHYES  16458 37237 1.084 0.022 THPR 424 Vif —TKLTEDRWNKPQKTK  10319 36434 1.137 0.014 GHR *PR values in bold indicatePR >1, i.e. OLP-responses seen more frequently in individuals withreduced viral loads.

In order to build a continuous immunogen sequence, the 26 OLPs werealigned and assembled to a total of 16 segments, ranging from 11-78amino acids in length. The precise starting and end positions of thesesegments were based on analyzing residues in up and down-steam of theidentified 26 OLPs and was based on a number of considerations that wereapplied to the different flanking sites. These considerations included:

1) OLP immunogenicity data

2) Conserved region reactivity data

3) Extension or chopping segments for inclusion/exclusion of good or badknown epitopes

4) CD4 epitope coverage

5) HLA coverage

6) Sequence variability (2010 consensus and HBX2 defined epitopes)

7) Multivariate OLP analyses

8) Creation of new epitope/self epitope

9) Maintenance of natural sequence though not included beneficial OLP

10) Introduction of changes to avoid epitope recognition and

11) Avoid forbidden residues (G, P, E, D, Q, N, T, S or C)

This protocol resulted in the design of SEQ ID NO: 1 to SEQ ID NO: 16 aspotential immunogens.

2. Vectors

Sequences SEQ ID NO: 1 to SEQ ID NO: 16 were linked with single, dual ortriple alanine amino acids between segments to ensure optimal processingand to avoid premature epitope digestion.

Then, the linked segments were used as HIV T cell immunogen sequencesfor inclusion in DNA and MVA vectors. For the delivery of the immunogensusing either soluble peptides only or in combination with heat shockproteins, shorter overlapping peptides (median length 23 residues) weredesigned that span the 16 segments, not including the triple AAAlinkers. These OLPs were generated in a way that helped avoid forbiddenresidues at the C-terminal end (important for optimal epitopepresentation on HLA class I molecules. See SEQ ID NO: 17 to SEQ ID NO:45, January 2012). These overlapping peptides range in length from 11-27amino acids.

3. T Cell Immunogen

The T cell immunogen has been designed as a polypeptide and assembledfrom 16 segments of the HIV-1 genome of varying size (between 11 to 78aa) unified by triple alanine linkers. Description of the regionsincluded:

T cell immunogen HIV-1 Position SEQ segments Length protein (HXB2) IDNO: Seg-1 78 p17 17-94 1 Seg-2 14 p24 30-43 2 Seg-3 11 p24 61-71 3 Seg-460 p24  91-150 4 Seg-5 14 p24 164-177 5 Seg-6 15 p24 217-231 6 Seg-7 27p2p7p1p6 63-89 7 Seg-8 55 protease 45-99 8 Seg-9 17 RT 34-50 9 Seg-10 55RT 210-264 10 Seg-11 34 RT 309-342 11 Seg-12 34 Integrase 210-243 12Seg-13 17 Integrase 266-282 13 Seg-14 23 Vif 25-50 14 Seg-15 19 Vif166-184 15 Seg-16 13 Nef 56-68 16Total length: 529 (including A, AA or AAA linkers)4. Inclusion of a Leader Sequence

Signal peptides are generally highly hydrophobic amino acid sequences(15 to 60 amino acids long) of proteins that must cross throughmembranes to arrive at their functioning cellular location. By bindingto signal recognition particles, these sequences direct nascentprotein-ribosome complexes to a membrane where the protein is insertedduring translation. Signal peptides direct translational uptake of theprotein by various membranes (e.g. endoplasmic reticulum, mitochondria,chloroplast, peroxisome). Leader signal sequences on non-membraneproteins are ultimately removed by specific peptidases.

Some signal peptides used include MCP-3 chemokine, for promotingsecretion and attraction of antigen presenting cells; a catenin(CATE)-derived peptide for increased proteasomal degradation; and thelysosomal associated protein, LAMP1 for targeting the MHC IIcompartment. See Rosati M, et al., Proc. Natl. Acad. Sci. USA 2009;106:15831-15836.

In the present design, the signal peptide from GMCSF (granulocytemacrophage colony-stimulating factor) was introduced at theamino-terminus of the immunogen to enhance secretion of the immunogenfrom expressing cells, followed by a valine to increase stability. Thesequence of the GMCSF signal peptide is:

(SEQ ID NO: 46) MWLQSLLLLGTVACSIS5. Inclusion of a Tag for In-Vitro Expression Experiments

For the purpose of assessing expression in transfected cells, theimmunogen sequence first included a FLAG peptide on the C-terminalregion, before the stop codon, was:

(SEQ ID NO: 48) DYKDDDDKL

The FLAG system utilizes a short, hydrophilic 8-amino acid peptide,which is fused to the recombinant protein of interest. The FLAG peptideincludes the binding site for several highly specific ANTI-FLAGmonoclonal antibodies (M1, M2, M5; Sigma-Aldrich Corp., Saint Louis,Mo., US), which can be used to assess expression of the protein ofinterest on material from transfected cells.

Because of the small size of the FLAG peptide tag, it does not shieldother epitopes, domains, or alter the function, secretion, or transportof the fusion protein generally. This sequence was removed afterwardsfor the mice immunogenicity assay. The FLAG tag is removed from thefinal immunogen (298H) before immunization.

6. Description of the T Cell Immunogen

The T cell immunogen has the following sequence (SEQ ID NO: 49):

M W L Q S L L L L G T V A C S I S V (E K I R L R P G G K K K Y K L K H I V W A S RE L E R F A V N P G L L E T S E G C R Q I L G Q L Q P S L Q T G S E E L K S L Y N TV A T L Y C V H Q K I E V )_(S1) A A A (K A F S P E V I P M F S A L)_(S2) A A A (G H Q AA M Q M L K E)_(S3) A A A (I A P G Q M R E P R G S D I A G T T S T L Q E Q I G W M TN N P P I P V G E I Y K R W I I L G L N K I V R M Y S P T S I )_(S4) A A A (Y V D R F Y KT L R A E Q A )_(S5) A (A C Q G V G G P G H K A R V L)_(S6) A A A (C T E R Q A N F L G KI W P S H K G R P G N F L Q S R)_(S7) A A A (K M I G G I G G F I K V R Q Y D Q I L I E IC G H K A I G T V L V G P T P V N I I G R N L L T Q I G C T L N F)_(S8) A A A (L V E I CT E M E K E G K I S K I)_(S9) A A A (L R W G F T T P D K K H Q K E P P F L W M G Y EL H P D K W T V Q P I V L P E K D S W T V N D I Q K L V G K L )_(S10) A A A (I L K E PV H G V Y Y D P S K D L I A E I Q K Q G Q G Q W T Y Q I Y)_(S11) A A A (T K E L Q KQ I T K I Q N F R V Y Y R D S R D P L W K G P A K L L W)_(S12) A A A (K I I R D Y G KQ M A G D D C V A)_(S13) A A (V K H H M Y I S K K A K G W F Y R H H Y E S T H P R)_(S14)A A A (V T K L T E D R W N K P Q K T K G H R)_(S15) A A (A W L E A Q E E E E V G F)_(S16)D Y K D D D D K Lwherein,the GMCSF signal peptide is shown underlined, the valine immediatelyfollowing the signal sequence is highlighted, the single, dual or tripleA (AAA) linkers are shown in bold, the FLAG epitope (removed in thefinal construct for in-vivo studies) is shown in italics and thedifferent segments are shown in brackets as follows:

Segment number HIV polypeptide HIV gene (. . .)_(S1) p17 (Seg-1) (. ..)_(S2-6) P24 (Seg-2 to Seg-6) {close oversize brace} gag (. . .)_(S7)p2p7p1p6 (Seg-7) (. . .)_(S8) Prot (seg-8) (. . .) _(S9-11) RT (Seg-9 toSeg-11) {close oversize brace} pol (. . .) _(S12-13) Int (Seg-12 andSeg-13 (. . .) _(S14-15) Vif (Seg-14 and Seg-15) Vif _(S16) Nef(Seg-16)Nef7. Nucleotide Sequence Codon Optimization

The T cell immunogen sequence was translated into a RNA/codon-optimizednucleotide sequence to enhance expression and secretion (Mr. Gene GmbH,Regensburg, DE). Codon optimization was based on introducing multiplenucleotide changes to destroy the previously identified RNA processing,inhibitory and instability sequences in the mRNA without affecting theencoded protein. See Schwartz S, et al., J. Virol. 1992; 66(12):7176-7182. This process can also include the elimination of predictedsplice sites (score >0.4) from coding sequences by appropriate codonchanges, to minimize the possibility of splicing.

As a result of the nucleotide changes indicated above, the finalGC-content of the T cell immunogen was 63%. The complete codon-optimizednucleotide sequence of the immunogen is (SEQ ID NO: 50):

   1 ATGTGGCCTCC AGAGCCTGCT ACTCCIGGGG ACGGTGGCCT CAGCATCIC GGTCGAGAAG  61 ATCCGGCTGC GGCCAGGCGG AAAGAAGAAG TACAAGCTGA AGCACATCGT CTGGGCCTCG 121 AGGGAGCTGG AGCGGTTCGC GGTGAACCCG GGACTTCTGG AGACGTCGGA GGGGTGCAGG 181 CAGATCCTCG GCCAGCTGCA GCCCTCTCTG CAAACGGGGT CTGAGGAGCT GAAGAGCCTG 241 TACAACACGG TGGCGACCCT CTACTGCGTC CACCAGAAGA TCGAGGTGGC AGCGGCCAAG 301 GCGTTCTCGC CGGAGGTCAT CCCCATGTTC TCGGCGCTGG CAGCTGCCGG ACACCAGGCC 361 GCGATGCAGA TGCTGAAGGA GGCCGCTGCG ATCGCACCGG GCCAGATGAG GGAGCCACGC 421 GGTTCCGACA TCGCGGGAAC CACCTCGACG CTCCAGGAGC AGATCGGATG GATGACGAAC 481 AACCCGCCAA TCCCGGTCGG GGAGATCTAC AAGCGGTGGA TCATCCTCGG GCTGAACAAG 541 ATCGTCCGGA TGTACAGCCC GACGTCGATC GCTGCGGCAT ACGTTGACCG GTTCTACAAG 601 ACCCTGAGGG CCGAGCAGGC AGCGGCCTGC CAGGGGGTCG GTGGACCAGG GCACAAGGCC 661 CGAGTGCTCG CGGCCGCATG CACGGAGCGG CAGGCGAACT TCCTGGGGAA GATCTGGCCG 721 TCGCACAAGG GCCGACCGGG AAACTTCCTC CAGTCTCGCG CAGCGGCTAA GATGATCGGA 781 GGCATCGGAG GCTTCATCAA AGTCCGTCAG TACGACCAGA TCCTCATCGA GATCTGCGGG 841 CACAAGGCGA TCGGAACCGT GCTCGTCGGC CCAACGCCCG TGAACATCAT CGGCCGCAAC 901 CTGTTAACGC AGATCGGCTG CACCCTCAAC TTCGCCGCAC TAGTGGAGAT CTGCACGGAG 961 ATGGAGAAGG AGGGCAAGAT ATCGAAGATC GCGGCAGCTC TGAGGTGGGG CTTCACCACG1021 CCGGACAAGA AGCACCAGAA GGAGCCGCCA TTCCTGTGGA TGGGATACGA GCTGCACCCG1081 GACAAGTGGA CCGTGCAGCC CATCGTCCTG CCGGAGAAGG ACTCGTGGAC GGTGAACGAC1141 ATCCAGAAGC TCGTGGGGAA GCTGGCGGCA GCCATCCTCA AGGAGCCCGT CCACGGGGTG1201 TACTACGACC CCTCTAAGGA CCTGATCGCG GAGATCCAGA AGCAGGGGCA GGGTCAGTGG1261 ACCTACCAGA TCTACGCAGC AGCAACCAAG GAGCTGCAGA AGCAGATCAC GAAGATCCAG1321 AACTTCCGCG TATACTACCG CGACTCGCGG GACCCCCTGT GGAAGGGCCC TGCGAAGCTT1381 CTCTGGGCAG CCGCGAAGAT CATCCGGGAC TACGGCAAGC AGATGGCGGG CGACGACTGC1441 GTGGCCGCAG CGGTGAAGCA CCATATGTAC ATCTCGAAGA AGGCGAAGGG CTGGTTCTAC1501 AGACACCACT ACGAGTCCAC CCACCCCAGG GCAGCTGCGG TGACGAAGCT GACGGAGGAC1561 CGGTGGAACA AGCCCCAGAA GACGAAGGGT CACCGGGCGG CTGCATGGCT GGAGGCTCAG1621 GAGGAGGAGG AGGTGGGCTT CGATTACAAG GACGATGACG ACAAGCTGtg ataawherein the sequence encoding GMCSF signal peptide is underlined, thevaline codon immediately downstream of the sequence encoding the signalsequence is shown highlighted, the sequence encoding the immunogenicpolypeptide is shown in standard letters, the sequence encoding the Flagtag is shown in italics and the tga and taa stop codons are shown inlower case.8. Cloning Strategy

The codon-optimized T cell immunogen was cloned into the mammalianexpression plasmid BV5, which consists of a modified CMV basic plasmidbackbone optimized for growth in bacteria that harbors the humancytomegalovirus (CMV) promoter, the bovine growth hormone (BGH)polyadenylation site and the kanamycin resistance gene—lacking the Xhosite. The cloning steps were as follows:

1) In a first step, an amino acid change from Leu to Meth was introducedinto the synthesized T cell immunogen—one including the FLAG epitope atRT 41 position (segment 9) to cover one of the major antiretroviralresistance mutations site. The T cell immunogen gene (starting vector)was cloned in a spectomycin resistance harboring plasmid. APCR-generated segment covering the RT M41 change was inserted into the Tcell immunogen as SpeI/HindIII. Competent cells DH108B were used fortransformation and were grown on LB-spectomycin media. The resultingplasmid was named HIVACAT RT M41. Insertion of the point mutation wasconfirmed by PCR sequencing using sense and antisense primers coveringthe segment 9 sequence.

2) In a second step, the HIVACAT RT M41 gene was inserted to the BV5plasmid that lacks the Xho site in kanamycin resistance gene asSalI/EcoRI, by ligation of the vector and the gel purified digestedHIVACAT RT M41 fragment. Competent cells DH108B were used fortransformation and were grown in LB-Kan media. Resulting plasmid namewas 297H (GMCSF-HIVACAT-FLAG). Insertion of the gene was confirmed byrestriction digestion and PCR sequencing using sense (from the CMVpromoter) and antisense primers (from the polyA BGH region).

3) In a third step, the epitope for the FLAG tag was removed from the297H plasmid by BstEII-EcoRI digestion and insertion the annealedprimers 298H Plus and 298H Minus:

298HPlus (SEQ ID NO: 51)GTCACCGGGCGGCTGCATGGCTGGAGGCTCAGGAGGAGGAGGAGGTGGGC TTCtgataaG298H Minus: (SEQ ID NO: 52)aattCttatcaGAAGCCCACCTCCTCCTCCTCCTGAGCCTCCAGCCATGC AGCCGCCCG

The resulting plasmid was named 298H GMCSF-HIVACAT, accession number DSM25555). See FIG. 1. Removal of the FLAG tag was confirmed by PCRsequencing using antisense primers (from the polyA BGH region).

Example 1

In-Vitro Expression Studies

Several transient transfections were performed to assess expression,localization and stability of the HIVACAT T cell immunogen.

Briefly, 1×10⁶ human 293 cells in complete DMEM plus 10% fetal bovineserum (FBS) were plated on to 60 mm tissue culture dishes and allowed toadhere overnight. HEK 293 cells were transfected by CaPhosphate DNAco-precipitation with a total of 7 μg of DNA (100 ng or 250 ng of the297H GMCSF-HIVACAT-FLAG plasmid DNA, 50 ng of GFP expressing plasmidpFRED143 topped up to 7 μg with Bluescript DNA).

6 hours after transfection the medium were replaced with 3 ml of DMEMsupplemented with 2% of FCS. After 24 and 48 hrs the cells and thesupernatants were collected in 0.5×RIPA.

Protein expression was analyzed by Western immunoblots. 1/250 of thetotal of the cell extracts and supernatants were loaded. The proteinswere resolved by electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gels (Nu-Page Bis-Tris, NuPAGE, Invitrogen, LifeTechnologies Corp., Carlsbad, Calif., US) and transferred ontonitrocellulose membranes.

297H plasmid was detected upon probing the membranes with horseradishperoxidase-conjugated anti-FLAG monoclonal antibody (Sigma-AldrichCorp., Saint Louis, Mo., US) at a 1:3.000 dilution.

Bands were visualized using ECL. Membranes were imaged on a ChemiDocXRS+.

Positive controls were used and included plasmid DNA encoding for cladeB p55 Gag, which also harbored the FLAG tag.

Cell extracts from transient transfections using the 298H plasmid(encoding for the HIVACAT T cell immunogen without the FLAG-tag) wereprobed with human serum from an HIV-1 infected subject at a 1:3.000dilution followed by a horseradish peroxidase-conjugated human anti-IgG,dilution 1:10.000.

297H and 298H plasmids stably (same estimated amount at 24 h and 48 h)expressed the HIVACAT T cell immunogen construct, which was visualizedat the cell extract compartment. There was no evidence of secretion ofthe construct.

Example 2

Cellular Response in Mice

A stock of 1 ml (2 mg/ml) of 298H GMCSF-HIVACAT DNA was producedendofree for in vivo studies in mice.

Immunogenicity of the HIVACAT T cell immunogen was evaluated in 6-8weeks old female C57BL/6 mice (Charles River Labs, Inc., Frederick, Md.,US).

20 μg and 5 μg of DNA was delivered intramuscularly by electroporationusing the Inovio system (Inovio Pharmaceuticals, Inc., Blue Bell, Pa.,US) in the left and right quadriceps (20 μg/50 μl per dose, 25 μl persite) at week 0 and 4. Mice were sacrificed 2 weeks after the lastimmunization. Mice splenocytes and serum were harvested forimmunogenicity studies. Control DNAs used were:

1) 114H p55 gag clade B: expresses full gag protein;

2) 132H NTV: expresses a chimaeric protein of nef, tat and vif;

3) 133H pol: expresses full pol protein; and

4) BV4 CMV-kan-Basic: SHAM control, similar DNA plasmid backbone withoutany expressed transgene.

35 mice were used in the experiment, pooling 5 mice per group.Distribution of the immunization per group was as follows:

DNA/Site Groups Inocula number Delivery Dose (quadriceps) n 1 114 p55gag I.M. 20 μg 25 mL/site 5 clade B Inovio 2 114 p55 gag I.M. 20 μg each25 mL/site 5 clade B + 132H Inovio NTV + 133 pol 3 298H GMCSF- I.M. 20μg 25 mL/site 5 HIVACAT Inovio 4 114 p55 gag I.M. 5 μg 25 mL/site 5clade B Inovio 5 114 p55 gag I.M.  5 μg each 25 mL/site 5 clade B + 132HInovio NTV + 133 pol 6 298H GMCSF- I.M. 5 μg 25 mL/site 5 HIVACAT Inovio7 BV4 CMVKan- I.M. 20 μg 25 mL/site 5 (SHAM) Basic Inovio

Cellular immune responses were characterized on a first step usingintracellular cytokine staining (ICS) in pooled splenocytes (cells fromthe 5 mice belonging to group) and using a pool of overlapping peptidescovering all gag, pol, nef, tat and vif proteins.

Briefly, pooled isolated mouse splenocytes from each group of mice wereincubated at a density if 2×10⁶ cells/ml, in 1 ml co-culture overnight,in the presence of peptide pools (15-mers, overlapping by 11aa coveringclade B gag, consensus B pol and NL43 nef, tat and vif sequences, 1μg/ml each peptide, total of about 12 hours, 1 hour without Golgi stopto prevent cytokine secretion). Surface immunostaining was performedwith CD3-allophycocyanin-Cy7, CD4-PerCP, CD8-Pacific Blue (BDBiosciences, Inc., Franklin Lakes, N.J., US). Intracellular cytokinestaining was performed using interferon gamma-FITC antibody (BDBiosciences, Inc., Franklin Lakes, N.J., US) after permeabilization.

From the first immunogenicity analyses, both 20 μg and 5 μg of DNA inC57BL/6 mice did generate detectable interferon gamma −+ responses tofull gag, pol and nef-tat-vif peptide pools. See FIG. 2a . Distributionof CD4+ and CD8+ responses is shown. See FIG. 2 b.

At an individual mice level, responses were deconvoluted using frozensplenocytes stimulated with 8 pools of peptides to cover the proteinsubunits included in the immunogen in an interferon gamma ELISpot assay.

ELISpot assay was performed by using mouse interferon gamma ELISpot kit(ALP) (Mabtech AB, Stockholm, SE) following the manufacturer'sinstructions with minor modifications. For all assays, mice splenocyteswere added at an input cell number of 4×10⁵ cells/well in 140 μl ofRosewell Park Memorial Institute medium 1640 with 10% fetal bovine serumin 96-well polyvinylidene plates (Millipore Corp., Bedford, Mass., US)alone or with HIV-1-specific peptide pools (14 μg/ml final concentrationfor each peptide) for 16 hours at 37° C. in 5% CO₂. Eight pools ofpeptides, each containing between 2 and 12 peptides of 18 amino acidsbased on the 2001 consensus-B sequence were pooled into the differentprotein subunits (gag-p17, gag-p24, gag-p2p7p1p6, pol-RT, pol-protease,pol-integrase, vif and nef) spanning the segments included in theHIVACAT T cell immunogen. The HIV peptides pools used in mice immunizedwith DNAs expressing full gag, pol, nef, tat and vif proteins, consistedof 18-mers peptides with an overlap of 11 residues spanning the completegag (6 pools, 11 peptides/each), pol (8 pools, 16 or 17 peptides/each),nef (2 pools, 13 o 14 peptides/each), tat (1 pool, 12 peptides) and vif(2 pools, 12 peptides/each) proteins.

Concavalin A (Sigma-Aldrich Corp., Saint Louis, Mo., US), at 5 mg/ml,was used as a positive control. The plates were developed with one-step5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium (BCIP/NBT,Bio-Rad Laboratories, Inc., Irvine, Calif., US). The spots on the plateswere counted using an automated ELISPOT reader system (CTL AnalyzersLLC, Cleveland, Ohio, US) using ImmunoSpot software and the magnitude ofresponses was expressed as spot forming cells (SFC) per million inputsplenocytes. The threshold for positive responses was defined as atleast 5 spots per well and responses exceeding the “mean number of spotsin negative control wells plus 3 standard deviations of the negativecontrol wells” and “three times the mean of negative control wells”,whichever was higher.

1) Dominance of interferon gamma responses developed in mice immunizedwith plasmids encoding for the entire gag, pol, nef, tat and vifproteins was towards regions outside the HIVACAT T cell immunogencovered segments (median ratio of responses targeting HIVACAT immunogenregions/total gag+pol+nef+tat+vif was 0.26 (range 0.17-0.42)) and didnot differ among groups immunized with high dose (20 μg) or low dose (5μg) of DNA. See FIG. 3

2) Median breadth of responses to protein subunits included in theHIVACAT T cell immunogen sequence was 4 (range 2-5) in mice immunizedwith 20 μg of HIVACAT vs 2 responses (range 1-3) in mice immunized with20 μg of plasmids encoding for entire proteins (ns) with no significantdifferences in the magnitude of responses. Six out of the eight proteinsubunits were at least targeted once in the mice immunized with theHIVACAT T cell immunogen. See FIG. 4.

HIVACAT Mice making a Mice making a T cell immunogen Peptides/ response(groups response (groups segments HIV-1 protein Pool number poolGag-Pol-NTV) HIVACAT) Seg-1 gag-p17 HTI-pool1 10 0/10 3/10 Seg-2 gag-p24HTI-pool2 12 10/10  10/10  Seg-3 gag-p24 Seg-4 gag-p24 Seg-5 gag-p24Seg-6 gag-p24 Seg-7 gag-p2p7p1p6 HTI-pool3 3 0/10 0/10 Seg-8pol-protease HTI-pool4 6 4/10 7/10 Seg-9 pol-RT HTI-pool5 11 5/10 9/10Seg-10 pol-RT Seg-11 pol-RT Seg-12 pol-integrase HTI-pool6 4 0/10 0/10Seg-13 pol-integrase Seg-14 vif HTI-pool7 4 3/10 2/10 Seg-15 vif Seg-16nef HTI-pool8 2 0/10 1/10

4) Dominance of responses in mice immunized with plasmids encoding thefull proteins of gag, pol, nef, tat and vif was 89% driven mainlytowards gag, while in mice immunized with the HIVACAT T cell immunogenat high doses was more balanced to all protein components (gag, pol, vifand nef) contained in the immunogen. See FIG. 5.

Example 3

Humoral Response in Mice

Humoral responses were first analyzed in pooled mice sera. Bindingantibodies to p24, p37 and p55 were detected by western immunoblot byusing cell extracts from HEK 293 cells transfected with the 1 mg of gagexpression vectors separated on 12% SDS-Page and probing the membraneswith pooled sera from mice (at a 1:100 dilution). Antibody titers to gagp24 were measured by ELISA. Serial 4-fold dilutions of the pooled serumsamples were assessed and the optical absorbance at 450 nm wasdetermined (Advanced BioScience Lab, Inc., Kensington, Md., US). Thebinding titers were reported as the highest dilution scoring positivehaving a value higher than the average plus 3 standard deviationsobtained with control sera from the mice immunized with SHAM DNA.

a) From the first humoral immunogenicity analyses, the HIVACAT T cellimmunogen induced binding antibody responses to gag p55, p37 and p24detectable by Western blot in the group of mice immunized with 20 μg.See FIG. 6.

b) Binding antibodies to p24 were quantified by ELISA. The endpointtiters of gag-p24 specific binding antibody from the mice that receivedthe plasmids described were determined by ELISA from individual serial4-fold diluted pooled serum samples. In the high dose group of miceimmunized with HIVACAT T cell immunogen at a titre of 1:4,000 which werelower to the titers detected in mice immunized with the full gagconstruct. No binding antibodies to p24 were measurable in the low dosegroup. See FIG. 7a . At an individual mice level, in house developed gagp55 ELISA using the HIV-1IIIB pr55 gag recombinant protein (Cat. No.3276, NIH Reagent Program, Bethesda, Md., US) was performed with micesera at 1:100 dilution. Low levels of antibody were detectable in 2 outof 3 mice immunized with the high dose of the immunogen. See FIG. 7 b.

Example 4

Heterologous Prime/Boost In-Vivo Immunogenicity in Mice

Material and Methods

Preparation of pDNA-HIVACAT and MVA-HIVACAT Vaccines

The codon-optimized T cell immunogen was cloned into the mammalianexpression plasmid BV5, which consists of a modified CMV basic plasmidbackbone optimized for growth in bacteria that harbors the humancytomegalovirus (CMV) promoter, the bovine growth hormone (BGH)polyadenylation site and the kanamycin resistance gene—lacking the Xhosite. The plasmid DNA for mice immunizations was prepared using theEndo-Free Megaprep (Qiagen) and stored −80° C. until use.

A recombinant MVA expressing the HIVACAT gene was made as describedpreviously {Letourneau, 2007 #235; Nkolola, 2004 #321}. Briefly, chickenembryo fibroblast (CEF) cells grown in Dulbeco's Modified Eagle's Mediumsupplemented with 10% FBS, penicillin/streptomycin and glutamine (DMEM10) were infected with parental MVA at MOI 1 and transfected usingSuperfectin (Quiagen) with 3 ug of pDNA-HIVACAT carrying the.beta.-galactosidase gene as a marker. Two days later, the total viruswas harvested and used to re-infect CEF cells. MVA was subjected to fiveround of plaque purification, after which a master virus stock wasgrown, purified on a 36% sucrose cushion, tittered and stored at −80° C.until use.

In-Vivo Immunogenicity in C57BL/6 Mice.

For heterologous prime/boost in-vivo immunogenicity experiments in mice,groups of five 6- to 8-weeks-old female C57BL/6 (Harlan LaboratoriesLtd., Barcelona, Spain) were used. Mice were primed intramuscularly with100 μg of pDNA-HIVACAT (2 or 3 vaccinations) followed by a 10{circumflexover ( )}6 pfu of MVA-HIVACAT boost (groups: 2×DNA, 3×DNA, 2×DNA+1MVAand 3×DNA+1MVA respectively) All vaccinations were separated by threeweeks.

All mice were sacrificed two weeks after the last vaccination in eachexperiment. Mice splenocytes and serum were harvested for immunogenicitystudies. Spleens were removed and pressed individually through a cellstrainer (Falcon) using a 5-ml syringe rubber punger. Following rbclysis, splenocytes were washed and resuspended in RPMI 1640 supplementedwith 10% FCS, penicillin/streptomycin (R10) and frozen until use.

All animal procedures and care were approved by a local Ethical Comitte.

Overlapping Peptides and Distribution of Peptide Pools

To evaluate immunogenicity of the heterologous regimens were pDNA or MVAexpressing only the HIVACAT T-cell immunogen and to rule outimmunogenicity of the potential junctional epitopes an overlappingpeptide set of 147 peptides of 15 amino acids in length (overlapping by11 residues) spanning the entire HIVACAT T-cell immunogen (including theleader sequence and linkers regions) was newly synthesized using9-Fluorenylmethyloxycarbonyl (Fmoc)-chemistry. Peptides were distributedin 18 different pools, according to protein subunits and segments of theimmunogen (1 pool for the signal peptide sequence, n=4 peptides; 7 poolsfor Gag, n=8-11 peptides/each; 7 pools for Pol, n=5-11 peptides/each; 2pools for Vif, n=6-8 peptides/each and 1 pool for Nef, n=2 peptides)Results are presented grouped by IFNγ responses specific for the eightprotein subunits (Gag p17, Gag p24, Gag p2p7p1p6, Pol-Protease, Pol-RT,Pol-Integrase, Vif and Nef)

Murine INFγ ELISPOT Assay

ELISpot assay was performed by using mouse IFNγ ELISpot kit (ALP)(Mabtech AB, Stockholm, SE) following the manufacturer's instructionswith minor modifications. For all assays, frozen mice splenocytes werefirst thawed and rested for 5 h 37° C. in R10 before use. Cells wereadded at an input cell number of 4×10⁵ cells/well in 140 μl of R10 in96-well polyvinylidene plates (Millipore Corp., Bedford, Mass., US)alone or with HIV-1-specific peptide pools (14 μm/ml final concentrationfor each peptide) for 16 hours at 37° C. in 5% CO₂. Concavalin A(Sigma-Aldrich Corp., Saint Louis, Mo., US), at 5 mg/ml, was used as apositive control. The plates were developed with one-step5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium (BCIP/NBT,Bio-Rad Laboratories, Inc., Irvine, Calif., US). The spots on the plateswere counted using an automated ELISPOT reader system (CTL AnalyzersLLC, Cleveland, Ohio, US) using ImmunoSpot software and the magnitude ofresponses was expressed as spot forming cells (SFC) per million inputsplenocytes. The threshold for positive responses was defined as atleast 5 spots per well and responses exceeding the “mean number of spotsin negative control wells plus 3 standard deviations of the negativecontrol wells” and “three times the mean of negative control wells”,whichever was higher.

Results

In these experiments as no mice were immunized using plasmids encodingfor full proteins, a second set of overlapping peptides matching theexact immunogen sequence was synthesized and used for immunogenicitycomparisons. Three intramuscular (i.m.) immunisations with 100 μg ofpDNA-HIVACAT were able to induce frequencies of IFNγ responses in allmice that were comparable to the frequencies of IFNγ responded inducedby immunisations with the electroporation Inovio system. However, twopDNA i.m. vaccinations were found to be immunogenic in only threeanimals (60%) compared to 100% of animals inducing a responses afterthree pDNA i.m. immunizations. Interestingly, MVA-HIVACAT vaccine wasable to boost responses both in breadth and magnitude, (FIG. 8B) in thetwo groups analyzed, but did just significantly increase the magnitudeof responses when mice had previously been primed with three doses ofpDNA-HIVACAT (FIGS. 8B and 8C). As seen in the previous EP experiments,a balanced and broad response to most of all the protein-subunitsincluded in the immunogen was observed in all animals, without a clearpattern of dominance among them. No nef or gag-p15 specific responseswere detected in the studied mice (FIG. 8D).

While the invention is described in some detail for purposes of clarityand understanding, it will be appreciated by one skilled in the art froma reading of this disclosure that various changes in form and detail canbe made without departing from the true scope of the invention andappended claims.

All publications mentioned hereinabove are hereby incorporated in theirentirety by reference.

The invention claimed is:
 1. A method of treating an HIV infection or adisease associated with an HIV infection in a subject in need thereof,the method comprising sequentially administering to the subject (1) anucleic acid encoding a first immunogenic polypeptide and (2) a secondimmunogenic polypeptide or a nucleic acid encoding a second immunogenicpolypeptide, wherein the first immunogenic polypeptide comprises thesequences of SEQ ID NOs:1-16 adjoined by amino acid linkers, wherein thesecond immunogenic polypeptide comprises the sequences of SEQ IDNOs:1-16 adjoined by amino acid linkers, wherein the nucleic acidencoding the first immunogenic polypeptide is administered to thesubject as part of an expression cassette, an expression vector, avirus, or a cell, wherein the nucleic acid encoding the secondimmunogenic polypeptide is administered to the subject as part of anexpression cassette, an expression vector, a virus, or a cell, andwherein (1) the nucleic acid encoding the first immunogenic polypeptideand (2) the second immunogenic polypeptide or the nucleic acid encodingthe second immunogenic polypeptide are administered in amounts effectivefor eliciting an immune response against HIV in the subject.
 2. Themethod of claim 1, wherein the subject is a human subject, and wherein(1) the nucleic acid encoding the first immunogenic polypeptide and (2)the second immunogenic polypeptide or the nucleic acid encoding thesecond immunogenic polypeptide are administered to the human subject totreat AIDS, ARC, or an HIV opportunistic disease.
 3. The method of claim1, wherein the amino acid linkers are single, dual, or triple alaninelinkers, and wherein the linkers result in the formation of an AAAsequence in the junction region between adjoining sequences.
 4. Themethod of claim 3, wherein the first and second immunogenic polypeptideshave the sequence of SEQ ID NO:99.
 5. The method of claim 1, wherein thenucleic acids encoding the first and second immunogenic polypeptideshave the sequence of SEQ ID NO:100.